DESCRIPTION (adapted from the application) The objectives of this application are to test hypotheses related to the importance of endocytic pathway abnormalities in amyloid precursor protein (APP) processing leading to increased b-amyloid protein production in sporadic Alzheimer's disease (AD) and the accentuation of these processes by genetic risk factors that lead to familial AD (FAD). The studies carried out thus far have shown increased activation of the endosomal-lysosomal system, including the endocytic pathway in sporadic AD. These studies suggest that the abnormal endosomal-lysosomal system activation and increased levels of lysosomal hydrolases lead to a continuum of progressive disturbances that are spatially and temporally associated with b-amyloid protein deposition and senile plaque formation. Recently, it has been found that abnormalities of the early endocytic pathway in sporadic AD is further influenced by certain genetic factors for AD (ApoE4 genotype), implying a mechanistic link between endosomal-lysosomal system alterations, genetic risk factors and b-amyloidogenesis. The progressive deposition of b-amyloid is a hallmark neuropathological feature of both sporadic and familial forms of AD and appears to be a necessary factor in AD pathogenesis; elucidation of the cellular pathways leading to its production and extracellular fate is a compelling strategy for identifying appropriate pharmacological intervention for AD. The specific aims of this application are: (i) to determine whether or not AD risk factors and genetic causes of AD accentuate endocytic abnormalities; (ii) to identify potential b-amyloid protein generating secretases that are present in increased amounts in early endosomes and to characterize trafficking mechanisms underlying enhanced delivery of these enzymes to early endosomes; (iii) to seek evidence for aberrant APP processing and b-amyloid generation within early endosomes from AD brains; and (iv) to use genetic manipulation to model endocytic and hydrolase abnormalities in cultured cells and prove predicted relationships to b-amyloid generation. The research design and methods proposed use immunological, cellular, and molecular approaches in vivo and in vitro to test the hypothesis that genetic risk factors for AD enhances b-amyloidogenesis by upregulating endocytosis, enzyme delivery to early endosomes, and accentuating other aspects of endocytic function in neurons. The applicant proposes to use genetically engineered cell models and fibroblast lines from sporadic AD and FAD individuals after further characterizing how primary genetic factors influence the nature of the cellular disturbances in endosomal-lysosomal system function in AD brain and will test the prediction that certain risk factors for AD will increase endosomal-lysosomal disturbances beyond what is seen in sporadic AD.